Dr. Yankelevich’s project title is “Development of Optimal Production, Cryopreservation, and Recovery of Mature Autologous Dendritic Cells in Preparation for a Phase I Study of Dendritic Cell-Based Vaccine in Children with Recurrent High Glade Gliomas.”
In spite of multimodal therapy, including maximal safe neurosurgical resection followed by radio-chemotherapy and maintenance chemotherapy, malignant or high-grade gliomas (HGG) continue to have a dismal prognosis both in children and adults. The preclinical, and the early clinical evidence have demonstrated a therapeutic potential of (DC)-based immunotherapy for HGG. Vaccination with tumor antigen-loaded DC’s generated in vitro has been shown to elicit antitumoral CTL responses responses in vivo and to induce tumor regression in some patients with gliomas. One of the critical steps in the DC-based immunotherapy is the ability to cryopreserve and subsequently revive sufficient numbers of functionally active clinical grade DC for sequential vaccination. DMSO is the most commonly used cryoprotectant with majority of DC studies using it at the concentrations of 10% or higher. However, DMSO at such concentration is toxic to cells, so it can negatively affect cell survival and function. Our preliminary results and recent literature reports suggest that using 5% DMSO vs. 10% DMSO may result in a better preservation of cells. The other advantage of using the lower concentration of DMSO is that it reduces the exposure to the patient. It will also enable us to reduce or avoid further manipulations with DC after thawing.
The other important step in the in the production of DC-vaccine is the antigen loading. Although DC’s loaded with many different preparations of antigens have been used as therapeutic vaccines, the optimal antigen loading method has not been determined. Previous studies demonstrated that dendritic cells loaded with whole tumor lysates, apoptotic tumor cells, or electroporated with whole tumor derived mRNA were able to process and present tumor tumor-associated antigens. Here we are planning to study the effects of different antigen-loading methods on dendritic cells phenotype, functional characteristics, and capacity to induce T-cell responses in vitro.
To test our hypothesis that optimized cryopreservation will improve DC viability and function we will first compare viability, phenotype, and functional features of mature DC cryopreserved with freezing medium containing 95% dextran-40 and 5% DMSO vs. freezing medium containing 12% DMSO and 44% AB serum. We will also examine whether Dextran-40 improves cellular penetration by DMSO as suggested by a recent report. After thawing, viability will be assessed by trypan blue exclusion, proportion of apoptotic cells will be tested by Annexin V FACS analysis, surface expression of molecules indicating mature status of DC will be tested by FACS analysis. In order to test the migratory properties cryopreserved and loaded DC migration assays will be performed. Finally, we will test the capacity of cryopreserved DC to induce the generation cytoxic T-cells against antigen-loaded DC’s in vitro with INF-gamma ELISPOT assay.
To test our second hypothesis that activation and maturation status of antigen-loaded dendritic cells as well as their T-cell responses inducing capacity are affected by the method of antigen loading we will load monocyte-derived DC’s from healthy volunteers using whole glioblastoma cell line lysate, glioblastoma line apoptopic cells, or glioblastoma cell line derived mRNA. We will study and compare phenotypical and functional characteristics of antigen-loaded DCs. T-cell responses against antigen-loaded DC’s will be measured by INF-gamma ELISPOT assay after one week of in vitro priming of autologous T-cells. Unloaded DC’s will serve as a control.
Results of this project will directly inform and lead to initiation of a phase I clinical trial of DC-based vaccine in children with recurrent HGG.